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Asian Pacificjournal of Tropical Medicine (2014)456-461

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Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

doi: 1O.1016/S1995-7645(14)60074-2

Synergistic effects of ethnomedicinal plants of Apocynaceae family andantibiotics against clinical isolates ofAcinetobacter baumannii

Sasitorn Chusri1,2*, Thanyaluck Siriyong, Pinanong N a- Phatthalung", Supayang PiyawanVoravuthikunchai2

,3

1FacultyofTraditionalThai Medicine, PrinceofSongkla University, Hat Yai, Songkhla 90112, Thailand2Natural ProductResearch Center ofExcellence, PrinceofSongkla University, Hat Yai, Songkhla 90112, Thailand3Department ofMicrobiology, FacultyofScience, PrinceofSongkla University, Hat Yai, Songkhla 90112, Thailand

ARTICLE INFO

Article history:Received 24 February 2014Received in revisedfmm 15March 2014Accepted 15April2014Available online 20June2014

Keywords:Acinetobacter baumanniiAntibacterial activityResistance modifying agentsApocynaceaeCombinationSynergistic effect

ABSTRACT

Objective: To investigate the efficacy of 17 ethnomedicinal plants belonging to Apocynaceaefamily used in combination with 16 conventional antibiotics against non-multidrug resistant-,multidrug resistant (MDR)-, and extensive drug resistant (XDR) Acinetobacter baumannii(A. baumanniii. Methods: Antibacterial activity and resistance modifying ability of 272combinations were determined by growth inhibition assays and further confirmed by time-killassay. Results: Among the combinations of the antibiotics with Apocynaceae ethanol extractson this pathogen, 15 (5%) had synergistic effects, 23 (8%) had partial synergistic effects and 234(86%) had no effects. Synergistic activity was observed mostly when the Apocynaceae extractswere combined with rifampicin or cefazolin. Interestingly, 10 out of 17 combinations betweenthe extracts and rifampicin displayed synergistic or partial synergistic behaviors. Holarrhenaantidysenterica extract was additionally tested to restore rifampicin activity against clinicalisolates of MDR and XDRA. baumannii. With respect to total or partial synergy, 70%was XDRA. baumannii isolates and 66% was MDRA. baumannii isolates. Conclusions: Holarrhenaantidysenterica extract clearly demonstrated the ability to restore rifampicin activity against bothA. baumannii ATCCI9606 and clinically isolated A. baumannii. Additional studies examining itsactive principles as well as mechanisms of actions such as the effects on efflux pumps and outermembrane permeability alterations are recommended.

1. Introduction

Increasing prevalence of multidrug resistant (MDR)bacteria and limited treatment options have necessitatedthe discovery of new antibacterial and resistance modifyingagents. Resistance modifying agents (RMAs) are compoundswhich potentiate the activity of an antibiotic against aresistant strain and may also target and inhibit MDRmeohanismsltl. An application of a RMA with a conventionalantibiotic is well accepted. Augmentin® is an important

·Corresponding author: Dr. Sasitorn Chusri, Faculty of Traditional Thai Medicine,Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.

E-mail: sasitom.chu@Psu.ac.thFoundation project: This work was supported by the Thailand Research Fund

(BRG 5580015, Fiscal year 2012-2014). Miss Pinaoong Na-Phatthalung aod MissThanyaluck Siriyong are funded by Natural Product Research Center of ExcellencePostgraduate Scholarship.

example which uses a combination of amoxicillin and amicrobial-derived beta-lactamase inhibitor as a RMA(clavulanatejlzl, Recent experiments have additionallydemonstrated that molecules capable of blocking theaction of efflux pumps have the potential to circumventantimicrobial resistancelsl, Stermitz et al reported for thefirst time the synergistic effect of a plant-derived ineffectiveantibacterial agent, berberine and a multi drug resistancepump inhibitor, 5'-methoxyhydnocarpin produced byBerberis species against S. aureus[4]. Furthermore, severalplant-derived alkaloids and polyphenols such as reserpine,quinine, harmaline, piperine, epigallocatechin gallate,tellimagrandin I, and rugosin B have been demonstrated toact as efflux pump inhibitors for Gram positive pathogen[5J.Recently, we have demonstrated that Holarrhenaantidysenterica (Linn) Wall. (Apocynaceae) possessed a

Sasitorn Chusri et al./Asian Pacific Journal of Tropical Medicine (2014)456-461 457

Table 1Medicinal properties and extraction yields of ethanol extracts of selected Apocynaceae ethnomedicinal plants.Medicinal plants (Plant parts) Medicinal properties Extraction yield (%; w/w)Adenium obesum (Forssk.) Roem. & Schult. (Leaves) Anti-cancer activity[20] 1.85Allamanda cathartica L. (Flowers) Treating malaria and jaundice[21] 4.79Alstonia macrophylla Wall. (Bark) Body tonic and anti-fever agents[22] 2.76Alstonia scholaris (L.) R.Br. (Bark) Treating asthma and cardiac[23] 4.43 Alyxia reinwardtii BL. var. lucida Markgr. (Branch) Antioxidant activity[24] 4.51Carissa spinarum L. (Branch) Wound healing activity[25] 3.50Catharanthus roseus L. (Branch) Used for treating cancers[26] 6.14Cerbera manghas L. (Bark) Anti-cancer activity[27] 12.20Cerbera odollam Gaertn. (Bark) Anti-cancer activity[28] 15.46Holarrhena antidysenterica (L.) Wall. (Bark) Antibacterial activity[29] 2.72Holarrhena curtisii King & Gamble (Branch) Leishmanicidal activities[30] 2.51Nerium oleander L. (Branch) Treating skin diseases[31] 4.52Plumeria obtusa (Bark) Treating skin diseases[32] 6.75Plumeria rubra L. (Bark) Antibacterial activity[33] 7.52Rauvolfia serpentina (L.) Benth. ex Kurz (Root) Antibacterial activity[34] 1.78Thevetia peruviana (Pers.) K. (Bark) Antidiarrhoeal and antimicrobial activities[35] 11.66Wrightia tomentosa Roem. & Schult. (Branch) Antibacterial activity[36] 2.75

Sasitorn Chusri et al.lAsian Pacific Journal of Tropical Medicine (2014)456-461 457

remarkable RMA ability in combination with novobiocinagainst Acinetobacter baumannii (A. baumannii) ATCC19606[6].

To our knowledge, there is no report on the RMA activity ofother ethnomedicinal plants from the family Apocynaceaeas well as relatively few studies have been carried out toevaluate RMA activities of plant-derived compounds on A.baumannii. Therefore, this study was aimed to investigatethe RMA activity of medicinal plants belonging to the familyApocynaceae in combination with conventional antibioticsagainst A. baumannii ATCC 19606and a collection of clinicalA. baumannii isolates.

2. Materials and methods

2.1. Bacterial strains and culture condition

Clinically isolated A. baumannii isolates were obtainedfrom Songklanakarin Hospital from pus (n=I), blood (n=2),sputum (n=5), body fluid (n=4), and urine (n=7) samples ofinfected patients. A. baumannii ATCC 19606 was employedin this study as a quality control strain. The strains werecultured on tryptic soy agar (TSA) (Difco Laboratories,Detroit, MI) and incubated at 37 ·C overnight. Colonies fromthe plates were grown in Mueller Hinton broth (MHB) (DifcoLaboratories, Detroit, MI) at 37 "Cfor 18-24 h and adjusted toMcFarland standards No. 0.5. The suspensions were furtherdiluted with MHB to obtain inocula containing lx106

CFU/mL.

Susceptibility test was performed by the disk diffusionmethod according to Clinical and Laboratory StandardInstitute (CLSI) recommendations[7J. MDR phenotypeswere defined as isolates resistant to at least three differentantimicrobial classes and the isolates resistant to all testedagents were classified as extensive drug resistant (XDR)phenotypeslsl,

2.2. Medicinal plant materials and extraction

Seventeen selected plant species belonging to theApocynaceae family were selected based on their potentialuse in folk medicine for treatments of diseases, or knownto have antimicrobial activities as described in Table 1.

The medicinal plants were purchased from medicinalherb retailers in Songkhla, Thailand and authenticatedby a taxonomist, Dr. Katesarin Maneenoon and voucherspecimens were deposited at the Faculty of Traditional ThaiMedicine, Prince of Songkla University, Hat Yai, Songkhla,Thailand. The samples were washed with distilled waterand dried at 60 "C overnight. Ground plant material (100 g)

was macerated with 95% (v/v) ethanol (500 mL) for 7 daysat room temperature. After filtrations through a WhatmanNo. 1 paper, the filtrates were concentrated using a rotatoryevaporator, and kept at 55 ·C until they were completelydried. Yields (%; w/w) of each extracts were calculated asthe ratio of the weight of the extract to the weight of theherb powder. A stock solution (200 mglmL) was preparedby dissolving the dried extract in dimethylsulfoxide (DMSO)(Merck, Germany).

2.3. Resistant modifying ability ofmedicinal plant extracts

Intrinsic anti-A. baumannii ATCC19606 activitiesof the Apocynaceae extracts and a panel of selectedantibiotics consisting of cell wall inhibitors (penicillin,oxacillin, ampicillin, imipenem, cefazolin, ceftazidime,and vancomycin), protein synthesis inhibitors (amikacin,gentamicin, streptomycin, fusidic acid, erythromycin, andtetracycline), DNA synthesis inhibitors (novobiocin andciprofloxacin), and RNA synthesis inhibitors (rifampicin)were determined by growth inhibition assays as previouslydescribedlsl, Briefly, the culture, containing ixio'CFU/mL (100 f.1. L) was inoculated into a 96-well microtiter

Table 1Medicinal properties and extraction yields of ethanol extracts of selected Apocynaceae ethnomedicinal plants.

Medicinal plants (plant parts)

Adenium obesum(Forssk.) Roem. & Schult. (Leaves)

Allamanda catharticaL. (Flowers)

AlstoniamacrophyllaWall. (Bark)

Alstoniascholaris (L.)R.Br. (Bark)

Alyxia reinwardtii BL. var. lucida Markgr. (Branch)

Carissa spinarumL. (Branch)

Catharanthusroseus L. (Branch)

Cerbera manghas L. (Bark)

Cerbera odollam Gaertn. (Bark)

Holarrhena antidysenterica (L.)Wall. (Bark)

Holarrhena curtisiiKing & Gamble (Branch)

NeriumoleanderL. (Branch)

Plumeria obtusa(Bark)

Plumeria rubraL. (Bark)

Rauvoljia serpentina (L.) Benth. ex Kurz (Root)

Theoetia peruviana (Pers.) K. (Bark)

WrightiatomentosaRoom. & Schult. (Branch)

Medicinal properties

Anti-cancer activity[20]

Treating malaria and jaundice[21]

Body tonic and anti-fever agents[22]

Treating asthma and cardiac[23]

Antioxidant activity[24]

Wound healing activity[25]

Used for treating cancers[26]

Anti-cancer activity[27]

Anti-cancer activity[28]

Antibacterial activity[29]

Leishmanicidal activities[30]

Treating skin diseases[31]

Treating skin diseases[32]

Antibacterial activity[33]

Antibacterial activity[34]

Antidiarrhoeal and antimicrobial activities[35]

Antibacterial activity[36]

Extraction yield (%;w/w)

1.854.792.764.434.513.506.14

12.2015.462.722.514.526.757.521.78

11.662.75

Sasitorn Chusri et al./Asian Pacific Journal of Tropical Medicine (2014)456-461458

Table 2 Intrinsic anti-Acinetobacter activitya of Apocynaceae ethnomedicinal plants.Bacterial growth inhibition (%)b No. of Apocynaceae (Plant species) 0-25 3 (Allamanda cathartica; Cerbera manghus; Thevetia peruviana)26-49 8 (Adenium obesum; Catharanthus roseus; Holarrhena antidysenterica; Holarrhena curtisii; Nerium oleander;

Plumeria obtusa; Plumeria rubra; Wrightia pubescens)50-75 4 (Alstonia scholaris; Alyxia reinwardtii; Cerbera odollam; Rauvolfia serpentine)76-100 2 (Alstonia macrophylla; Carissa spinarum)

aAn antibacterial activity of phytochemicals is considered to be significant if MIC values are below 100 毺g/mL for crude extract and 10 毺g/mL for pure compounds[37]. bThe percentage inhibition of bacterial growth was calculated by using the equation:Bacterial growth inhibition (%) = (ODcontrol-ODtest) /ODcontrol ×100. Where, ODcontrol represents the optical density at 620 nm of the control culture in MHB containing 1% (v/v) DMSO, ODtest represents the optical density at 620 nm of the culture in MHB containing 1 mg/mL of the ethanol extract. The ODtest of respective blanks having only the extract was subtracted to give the final ODtest.

458 Sasitorn Chusri et al./Asian Pacific Journal of Tropical Medicine (2014)456-461

plate containing 50 fL L of the extract (1 000 fL g/ml.) orthe antibiotic and 50 fL L of MHB. The antibiotics werepurchased from Becton Dickinson Microbiology Systems(Sparks, MD, USA), Difco (Detroit,MI,USA) or made using thelaboratory collection of antibiotics.

The intrinsic antibacterial activity was exhibited as thepercentage of growth inhibition (GI) after incubation at 37 ·Cfor 18 h and calculated from the following equation:

GI (%) = (ODcontrol-ODte,J/ODcontrolxl00. (1)

where, ODcontrol is optical density (00) 620 nm of bacteriaculture in MHB supplemented with 1% (v/v) DMSO as apositive control and ODte,t is 00 620 nm of the bacterialculture in MHB supplemented with the tested agent. TheODte,t of respective blanks having only the extract wassubtracted to give the final ODte,t. GIA and GIE represent thepercentage inhibition of bacterial growth of the antibioticand extract, respectively.

Resistance modifying ability of each extract was observedby adding of 50 fL L of the tested extract into the testedplate supplemented with the antibiotic instead of MHB. Thisbiological activity was exhibited as the percentage of growthinhibition as well but calculated from the followingequation:

%Growth inhibition of the combination (Glc) =ODcontrol­ODtesJIODcontrolx100. (2)

where, ODcontrol is 00 620 nm of the positive control cultureand ODte,t is 00 620 nm of the bacterial culture in MHBsupplemented with the extract in combination with theantibiotic.

The interpretation of the combination was classified assynergism when GIc/GIA and GIc/GIE ratios were ~2.0,

partial synergism when 1.5:S;the ratios<2.0, and no effectwhen the ratios <1.5. Ellagic acid at 40 fL mollL wasincluded as a positive control RMA in combination witherythromycin, novobiocin, and rifampicin against A.baumannii ATCCI9606.

The efficacy of combination therapy of the promisingmedicinal plants in combination with the antibiotics wasadditionally determined against 19 clinically isolated A.baumannii isolates using the growth inhibition assay asdescribed above and further confirmed by a time-kill assay.

3. Results

In this present investigation, the growth inhibition assaywas employed to develop another approach for combating A.baumannii infections using medicinal plants to potentiatethe activity of antibiotics. Independently, 15 out of 17 testedethanol extracts at concentration of 1 000 fL g/mL displayedlow inherent anti-A. baumannii activity (% of bacterialgrowth inhibition was less than 75%) (Table 2). Only Alstoniamacrophylla and Carissa spinarum which completelyinhibited the bacterial growth at this concentrationpossessed moderate antibacterial activity.

From 272 combinations tested between 17 medicinalplants and 16 antibiotics, 15 (5%) showed synergism, 23(8%) had partial synergistic interaction, and 234 (86%) hadno effect. Synergistic activity was observed mostly whenthe Apocynaceae extracts were combined with rifampicinor cefazolin against A. baumannii ATCC19606. Synergisticbehaviors were displayed in cefazolin in combinationwith Alstonia scholaris, Cerbera odollam, Holarrhenaantidysenterica, Nerium oleander, or Thevetia peruviana orrifampicin in combination with Adenium obesum,Holarrhenaantidysenterica, Plumeria obtuse, Thevetia peruviana, orWrightiapubescens (Table 3).

The ability of a representative effective resistancemodifier, Holarrhena antidysenterica to potentiate theantibacterial activity of rifampicin against clinically isolatedA. baumannii was additionally evaluated to explore thepotential of developing a promising RMA (Table 4). Theinteraction between the ethanol extract and rifampicin wassynergistic and partially synergistic in 8 (42.1%) and 3 (15.8%)isolates of A. baumannii tested, respectively. With respectto total or partial synergy, 70%, 66%, and 33% of the isolateswere XDRA. baumannii, MORA. baumannii, and non-MRDA. baumannii, respectively.

The synergistic effect of this combination was furtherconfirmed by time-kill assay as illustrated in Figure1. At the tested concentration, the extract exhibited noantibacterial potencies, but it was shown to be a powerfulRMA in combination with rifampicin against A. baumanniiATCC 19606, non-MDR A. baumannii, and XDR A.baumannii.

Table 2Intrinsic anti-Acinetobacter activity" of Apocynaceae ethnomedicinal plants.

Bacterial growth inhibition (%)b No. of Apocynaceae (Plant species)

0-25 3 (Allamanda cathartica; Cerbera manghus; Thevetia peruviana)

26-49 8 (Adenium obesum; Catharanthus roseus; Holarrhena antidysenterica; Holarrhena curtisii; Nerium oleander;Plumeria obtusa; Plumeria rubra; Wrightia pubescens)

50-75 4 (Alstonia scholaris; Alyxia reinwardtii; Cerbera odollam; Rauuolfia serpentine)76-100 2 (Alstonia macrophylla; Carissa spinarum)

"An antibacterial activity of phytochemicals is considered to be significant if MIC values are below 100 f1. g/mL for crude extract and 10 f1. g/mLfor pure compounds[37].bThe percentage inhibition of bacterial growth was calculated by using the equation:

Bacterial growth inhibition (%) =(OD"nt"l-OD",,) IOD"nt"l x 100. Where, OD"nt"l represents the optical density at 620 nm of the control culturein MHB containing 1% (v/v) DMSO, OD"" represents the optical density at 620 nm of the culture in MHB containing 1 mg/mL of the ethanolextract. The OD"" of respective blanks having only the extract was subtracted to give the final OD"".

Sasitorn Chusri et al./Asian Pacific Journal of Tropical Medicine (2014)456-461 459

Table 3Resistance modifying ability of Apocynaceae ethanol extracts in combination with selected antibiotics against Acinetobacter baumannii ATCC 19606.

Plant species Number of synergy or partial synergy interactions [Antibioticsa; (GIC:GIA/GIC:GIE)b]Synergyc Partial synergyc

Adenium obesum 1 RIF (2.8/2.0) 3 MEM (1.8/4.6), GEN (1.5/1.7), ERY (1.5/4.9)Allamanda cathartica 0 - 1 FUS (1.7/3.3)Alstonia macrophylla 0 - 0 -Alstonia scholaris 1 KZ (21.6/7.9) 0 -Alyxia reinwardtii 0 - 2 KZ (17.8/1.5), RIF (2.6/1.5)Catharanthus roseus 0 - 1 STR (1.8/22.0)Carissa spinarum 0 - 0 -Cerbera manghus 3 MEM (3.5/4.7), GEN (4.2/2.6), ERY (2.4/4.3) 2 AMP (1.7/3.6), RIF (4.7/1.8)Cerbera odollam 1 KZ (21.4/3.5) 5 PEN(2.4/1.9), OXA (2.2/1.9), AMP (2.3/1.7), VAN

(2.7/1.5), RIF (2.0/1.9)Holarrhena antidysenterica 3 KZ (21.4/3.5), STR (3.9/9.5), RIF (2.7/3.0) 0 -Holarrhena curtisii 0 - 1 KZ (11.9/1.6)Nerium oleander 1 KZ (15.7/2.7) 1 RIF (1.7/2.0)Plumeria obtusa 1 RIF (2.0/4.2) 0 -Plumeria rubra 0 - 2 GEN (1.6/2.3), RIF (1.7/1.5)Rauvolfia serpentin 0 - 0 -Spirolobium cambodianum 0 - 0 -Thevetia peruviana 3 KZ (21.1/18.4), FUS (2.2/2.3), RIF (2.1/6.2) 0 -Wrightia pubescens 1 RIF (2.0/2.1) 5 MEM (1.7/6.4), KZ (1.8/35.3), GEN (1.8/3.1), STR

(1.9/24.0), ERY (1.9/9.0),aPEN=Penicillin G; OXA=Oxacillin; AMP=Ampicillin; MEM=Meropenem; KZ=Cephazolin; VAN=Vancomycin; GEN=Gentamicin; STR=Streptomycin; FUS=Fusidic acid; ERY=Erythromycin; RIF=Rifampicin.bGIC represents the percentage inhibition of bacterial growth of tested antibiotics in combination with the extracts. GIA and GIE represent the percentage inhibition of bacterial growth of the antibiotics and the extracts, respectively. cCombinations were classified as synergistic effects if both GIC:GIA and GIC:GIE ratios were 曒2.0, partially synergetic effects if 1.5曑the ratios<2.0, indifferent effects if the ratios were <1.5.

0.550.500.450.400.350.300.250.200.150.100.050.00

-0.05

OD62

0 (M

ean依

SD)

A

0 2 4 8 16 18 Time (h)

ControlHANoRiNo+HARi+HA

0.550.500.450.400.350.300.250.200.150.100.050.00

-0.05

OD62

0 (M

ean依

SD)

B0.550.500.450.400.350.300.250.200.150.100.050.00

-0.05

OD62

0 (M

ean依

SD)

C

0 2 4 8 16 18 Time (h)

ControlHANoRiNo+HARi+HA

0 2 4 8 16 18 Time (h)

ControlHANoRiNo+HARi+HA

Figure 1. Time-kill activities of Holarrhena antidysenterica (HA; 125 毺g/mL), rifampicin (Ri; 1/4 MIC), novobiocin (No; 1/4 MIC), the combination of Holarrhena antidysenterica and rifampicin (Ri+HA), and the combination of Holarrhena antidysenterica and novobiocin (No+HA) againt Acinetobacter baumannii ATCC 19606 (A), non-MDR A. baumannii (B), and XDR A. baumannii (C). Minimun inhibitory concentrations of Ri and No against A. baumannii ATCC 19606, non-MDR A. baumannii, and XDR A. baumannii were 0.63 and 6.25, 5.00 and 25.00, and 1.25 and 6.25 毺g/mL, respectively.

Sasitorn Chusri et al.lAsian Pacific Journal of Tropical Medicine (2014)456-461 459

Table 3Resistance modifying ability of Apocynaceae ethanol extracts in combination with selected antibiotics against Acinetobacter baumannii ATCC19606.

Plant speciesNumber of synergy or partial synergy interactions [Antibiotics"; (GIc:GIA/GIc:GIE)b]

Synergy' Partial synergy'Adenium obesum

Allamanda cathartica

Alstonia macrophylla

Alstonia scholaris

Alyxia reinwardtii

Catharanthus roseus

Carissa spinarum

Cerbera manghus

Cerbera odollam

Holarrhena antidysenterica

Holarrhena curtisii

Nerium oleander

Plumeria obtusa

Plumeria rubra

Rauvoifia serpentui

Spirolobium cambodianum

Thevetia peruviana

Wrightia pubescens

1 RIF (2.8/2.0)o -o -1 KZ (21.617.9)o -o -o -3 MEM(3.5/4.7), GEN (4.2/2.6), ERY (2.4/4.3)1 KZ (21.4/3.5)

3 KZ (21.4/3.5), STR (3.9/9.5), RIF (2.7/3.0)o -1 KZ (15.7/2.7)1 RIF (2.0/4.2)o -o -o -3 KZ (21.1/18.4), FUS (2.2/2.3), RIF (2.1/6.2)1 RIF (2.0/2.1)

3 MEM(1.8/4.6), GEN (1.5/1.7), ERY (1.5/4.9)1 FUS (1.7/3.3)o -o -2 KZ (17.8/1.5), RIF (2.6/1.5)1 STR (1.8/22.0)o -2 AMP (1.7/3.6), RIF (4.7/1.8)5 PEN(2.411.9), OXA (2.211.9), AMP (2.311.7), VAN

(2.7/1.5), RIF (2.0/1.9)o -1 KZ (11.9/1.6)1 RIF (1.7/2.0)o -2 GEN (1.6/2.3), RIF (1.7/1.5)o -o -o -5 MEM (1.7/6.4), KZ (1.8/35.3), GEN (1.8/3.1), STR

(1.9/24.0), ERY (1.9/9.0),

apEN=Penicillin G; OXA=Oxacillin; AMP=Ampicillin; MEM=Meropenem; KZ=Cephazolin; VAN=Vancomycin; GEN=Gentamicin;STR=Streptomycin; FUS=Fusidic acid; ERY=Erythromycin; RIF=Rifampicin.bGIc represents the percentage inhibition of bacterial growth of tested antibiotics in combination with the extracts. GIAand GIE represent thepercentage inhibition of bacterial growth of the antibiotics and the extracts, respectively.'Combinations were classified as synergistic effects if both GIc:GIAand GIc:GIE ratios were ~2.0, partially synergetic effects if 1.5~the

ratios<2.0, indifferent effects if the ratios were <1.5.

18164 8Time (h)

2o

c0.550.50 ,

08::81

'II 0.35~ 0.30'

:E 0.25~ 0.20 '~ 0.15'6 0.10 '

0.0510.00 '

-0.05~--~-~------

- Control

:: ~~-- Ri." .. No+HA._. Ri+HA

16 182 4 8Time (h)

o

B0.550.500.45

- Control 00.40--HA 'II 0.35::N9 § 0.30'-'~~+HA :S 0.25·_··Ri+HA ~ 0.20

~ 0.156 0.10

0.050.00

-0.0518164 8Time (h)

2

A 0.550.50 '

08::8'II0.35 f~0.30 ·

68'~8 rJO:150 0.10.

0.050.00

-0.05 '~------,-------,-------,-----,----,-----

Figure 1. Time-kill activities of Holarrhena antidysenterica (HA; 125 IJ.. g/mL), rifampicin (Ri; 1/4 MIC), novobiocin (No; 1/4 MIC), thecombination of Holarrhena antidysenterica and rifampicin (Ri+HA), and the combination of Holarrhena antidysenterica and novobiocin (No+HA)againtAcinetobacter baumannii ATCC 19606 (A), non-MDRA. baumannii (8), and XDRA. baumannii (C). Minimun inhibitory concentrationsof Ri and No against A. baumannii ATCC 19606, non-MDR A. baumannii, and XDR A. baumannii were 0.63 and 6.25, 5.00 and 25.00, and1.25 and 6.25 IJ.. g/mL, respectively.

4. Discussion

Uses of rifampicin in combination with colistin have beenstudied for the treatment of MDR A. baumannii infections.Both in vitro studies and clinical studies were employedto recommend the safety and clinical effectiveness ofrifampicin in combination with colistin against thispathogen[lO-131. It was suggested that colistin probablycauses rapid permeabilization of the outer membrane, whichenhances penetration and activity of rifampicin. Similarly,plant-derived compounds that act as permeabilizers such as

coriander oil (Coriandrum sativum)[14I, geraniol tHelichrysumitalicum)[151, and [6]-dehydrogingerdione and [lO]-gingerol(Zingiber officinale)[161 have been shown to reduce theresistance of A. baumannii to other antibiotics. Even thoughantibacterial activity of Holarrhena antidysenterica and itsconstituents have been reported, there is to our knowledgeno published scientific literature of RMA activity onrifampicin of this plant or its constituents.

Rifampicin resistance in A. baumannii is related to thesynergistic interaction between modifications of antibioticpermeability, enzymatic modification by rifampicin

Sasitorn Chusri et al./Asian Pacific Journal of Tropical Medicine (2014)456-461460

Table 4Effects of Holarrhena antidysenterica ethanol extract (HA; 125 毺g/mL) as a resistant modifying agent for rifampicin (Ri; 0.5 毺g/mL) against clinically isolated A. baumannii.

MDR- A. baumannii isolates (Sources)a Antibiogram profileb GI (%)c in the presence ofGIC:GIA/GIC:GIE

d

Ri (0.5 毺g/mL) HA (125 毺g/mL) NPRC AB002 (BF) SRISRSSSSS 13.2依1.3 13.6依1.1 1.0/1.0NPRC AB003 (S) RRRRRRRRSR 40.8依1.4 35.7依1.0 2.5/2.9NPRC AB004 (U) SRISISSSSS 89.2依1.2 2.0依1.7 1.1/7.0NPRC AB005 (U) RRRRR-RSIS 19.2依0.6 -21.0依1.5 1.8/1.7NPRC AB010 (B) RRRRRRRRSR 30.7依1.8 35.5依3.7 1.6/1.4NPRC AB011 (U) RRRRRRRRIR 37.2依0.5 37.3依1.3 1.0/1.0NPRC AB013 (B) RRRRRRRRRR 17.8依2.6 37.9依2.9 4.8/2.3NPRC AB014 (U) SRRSR-SRIR 19.0依4.1 22.0依2.8 4.4/3.8NPRC AB015 (U) RRRRR-RRRR 25.3依0.7 23.0依1.4 2.1/2.3NPRC AB016 (U) SRRSRRSSSS 51.8依1.9 23.8依5.0 1.2/2.7NPRC AB017 (BF) RRRRRRRRRR 64.7依0.9 56.1依1.1 1.6/1.9NPRC AB018 (S) RRRRRRRRRR 32.8依2.4 34.8依2.0 2.0/1.9NPRC AB019 (S) RRRRRRRRRR 33.3依0.8 30.0依0.8 2.3/2.5NPRC AB021 (S) SRISR-SRSR 40.7依2.8 27.5依1.6 2.1/3.2NPRC AB022 (U) RRRRRRRRRR 18.1依1.6 39.2依1.1 2.1/0.4NPRC AB024 (BF) -RRR-R-R-- 57.9依0.9 27.8依1.9 1.8/3.7NPRC AB026 (S) RRRRRRRRSR 34.4依0.7 37.3依0.8 4.7/3.0NPRC AB028 (BF) RRRRRRRRIR 35.9依1.5 28.8依1.0 2.1/2.6NPRC AB029 (P) SRSSSSSSSS 17.4依4.8 27.5依3.8 1.4/8.7

aClinically isolated A. baumannii were obtained from pus (P), blood (B), sputum (S), body fluid (BF), and urine (U) samples of infected patients. bThe antibiogram profile is the susceptibility results for amikacin, ampicillin, cefotaxime, cefazolin, cefuroxime, ciprofloxacin, gentamicin, imipenem, cephoperazon/sulbactam, and meropenem.cGI represents the percentage inhibition of bacterial growth of the tested compounds. dCombinations were classified as synergistic effects (GIC:GIA and GIC:GIE ratios 曑2.0), partially synergetic effects (1.5曑GIC:GIA and GIC:GIE

ratios<2.0), indifferent effects (GIC:GIA and GIC:GIE ratios <1.5). GIA, GIE, and GIC represent the percentage inhibition of bacterial growth of Ri, HA, and the Ri+HA combination, respectively.

460 Sasitorn Chusri et al./Asian Pacific Journal of Tropical Medicine (2014)456-461

Table 4Effects of Holarrhena antidysenterica ethanol extract (HA; 125 f1. g/mL) as a resistant modifying agent for rifampicin (Ri; 0.5 f1. g/mL) againstclinically isolated A. baumannii.

MDR- A. baumannii isolates (Sources)" Antibiogramprofile"GI (%r in the presence of

Glc:GI)Glc:GIEdRi (0.5 f1. gimL) HA (125 f1. gimL)

NPRC AB002 (BF) SRISRSSSSS 13.2±1.3 13.6±1.1 1.0/1.0NPRC AB003 (S) RRRRRRRRSR 40.8±1.4 35.7±1.0 2.5/2.9NPRC AB004 (U) SRISISSSSS 89.2±1.2 2.0±1.7 1.117.0NPRC AB005 (U) RRRRR-RSIS 19.2±0.6 -21.0±1.5 1.8/1.7NPRC ABOIO (B) RRRRRRRRSR 30.7±1.8 35.5±3.7 1.6/1.4NPRC ABOlI (U) RRRRRRRRIR 37.2±0.5 37.3±1.3 1.0/1.0NPRC AB013 (B) RRRRRRRRRR 17.8±2.6 37.9±2.9 4.8/2.3NPRC AB0l4 (U) SRRSR-SRIR 19.0±4.1 22.0±2.8 4.4/3.8NPRC AB0l5 (U) RRRRR-RRRR 25.3±0.7 23.0±1.4 2.1/2.3NPRC AB0l6 (U) SRRSRRSSSS 51.8±1.9 23.8±5.0 1.2/2.7NPRC AB0l7 (BF) RRRRRRRRRR 64.7±0.9 56.1±1.1 1.6/1.9NPRC AB0l8 (S) RRRRRRRRRR 32.8±2.4 34.8±2.0 2.0/1.9NPRC AB0l9 (S) RRRRRRRRRR 33.3±0.8 30.0±0.8 2.3/2.5NPRC AB021 (S) SRISR-SRSR 40.7±2.8 27.5±1.6 2.1/3.2NPRC AB022 (U) RRRRRRRRRR 18.1±1.6 39.2±1.1 2.1/0.4NPRC AB024 (BF) -RRR-R-R-- 57.9±0.9 27.8±1.9 1.8/3.7NPRC AB026 (S) RRRRRRRRSR 34.4±0.7 37.3±0.8 4.7/3.0NPRC AB028 (BF) RRRRRRRRIR 35.9±1.5 28.8±1.0 2.1/2.6NPRC AB029 (P) SRSSSSSSSS 17.4±4.8 27.5±3.8 1.4/8.7

"Clinicallyisolated A. baumannii were obtained from pus (P), blood (B),sputum (S), body fluid (BF),and urine (U)samples of infected patients.bThe antibiogram profile is the susceptibility results for amikacin, ampicillin, cefotaxime, cefazolin, cefuroxime, ciprofloxacin, gentamicin,imipenem, cephoperazon/sulbactam, and meropenem.'GI represents the percentage inhibition of bacterial growthof the tested compounds.dCombinations were classified as synergistic effects (Glc:GIAand Glc:GIE ratios ~2.0), partially synergetic effects (1.5~Glc:GIA and Glc:GIEratios<2.0), indifferent effects (Glc:GIAand Glc:GIE ratios <1.5). GIA, GIE, and Glc represent the percentage inhibition of bacterial growthof Hi,HA, and the Ri+HA combination, respectively.

ADP-ribosyl-transferase (arr-2), or mutation in rpoB[17­

191. A recent finding by Giannouli et al proposed that thecombined treatment with colistin/rifampicin versus colistinalone were evident only in A. baumannii strains with nochromosomal mutations in RNA polymerase ~ -subunitrpoB target genelttl, Interestingly, phenylalanine arginine~ -naphthylamide (PA ~ N), an efflux pump inhibitor,

reduced the minimun inhibitory concentration ofrifampicin at 256 p. g/mL by approximately 30-fold inA. baumannii isolate that showed no mutation in the rpoBtarget genelttl.

The present results indicate that the ethanol extractof Holarrhena antidysenterica is a promising resistancemodifying agent for rifampicin against A. baumannii, due toits synergistic effect in combination with rifampicin againstboth A. baumannii ATCC19606 and clinically isolatedA. baumannii. The findings may lead to developmentof an effective alternative treatment in combating theantimicrobial resistance in A. baumannii. Therefore, themechanisms of action of this combination as well as theactive constituents of Holarrhena antidysenterica should befurther investigated.

Acknowledgments

This work was supported by the Thailand Research Fund(BRG 5580015, Fiscal year 2012-2014). Miss Pinanong Na-

Phatthalung and Miss Thanyaluck Siriyong are funded byNatural Product Research Center of Excellence PostgraduateScholarship. Weare thankful to Miss Stefania Vignotto forediting the manuscript.

Conflict of interest statement

We declare that we have no conflict of interest.

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